Hoskins et al. use a single-molecule fluorescence approach that dubbed “CoSMoS” (Co-localization Single Molecule Spectroscopy), originally developed in the Gelles lab by Larry Friedman and Johnson Chung, that is a powerful method to study the assembly and function of the complex macromolecular machines that perform a wide variety of biological functions. In this movie, shown 150x faster than real time, the comings and goings of many U1 spliceosome components on a surface-tethered pre-mRNA are shown as the appearance and disappearances of white spots. The white spots orginate from the fluorescence emission of specifically labeled U1 components upon excitation with a 532nm laser.

Pre-mRNAs are spliced in a complex cycle wherein the spliceosome assembles, is activated for catalysis, performs two transesterification reactions, and disassembles on every turnover. Steps between the isolatable intermediates depicted in this cycle involve the coordinated association and dissociation of many spliceosome components. A key finding by Hoskins et al. is that spliceosome assembly is reversible, and this is represented by the dashed arrows between the pre-mRNA, A, and B complexes.

The multi-wavelength, total internal reflection fluorescence (TIRF) microscope built by Larry Friedman and Johnson Chung in the Gelles laboratory uses lasers of different wavelengths to excite spectrally distinguishable fluorophores on various spliceosome components. Photo by Diane Katherine Hunt.

According to Hoskins, who will leave Brandeis to take up a faculty position in the Biochemistry Department at the University of Wisconsin, Madison

By far, the most challenging aspect of the project was determining two completely orthogonal methods for attaching fluorophores to endogenous spliceosomes in whole cell extract. Since these experiments are quantitative, we needed to find methods that give a very high degree of fluorophore incoporation and specificity (in other words, 10% labeling would not cut it!).

The novel part, for me, is that for decades spliceosome kinetics have been “off-limits” to enzymologists due to the complexity of the system. However, by developing the correct analytical tools, the spliceosome can be studied in detail usually reserved for enzymes orders of magnitude smaller.

Hoskins plans to continue these single molecule studies of the spliceosome in his new lab in Wisconsin and will be focusing on splice site selection and coupling of nuclear RNA processing events. He also aims to develop new methodologies for fluorescent labeling of ribonucleoproteins in vitro and in vivo.

Single particle electron microscopy reconstruction can be a powerful tool for determining the structure of large protein complexes. One limitation of the technique is the difficulty in coming up with specific labels for the protein that can be visualized with EM. In a new paper in RNA, postdoc Beth Stroupe and coworkers show that the use of the actin-nucleating protein Spire as a cloneable tag allows them to nucleate actin filaments that then “point” to the location of the tag in the complex seen in EM, and applied the technique to their studies of the C complex spliceosome.